Treating Inflammatory Disorders With Antibodies to the Alpha-7 Nicotinic Receptors

ABSTRACT

An antibody or an antigen binding fragment thereof which binds to mammalian α7 subunit of a nicotinic acetylcholine receptor or its functional variant and which is an agonist of said receptor or variant. Pharmaceutical compositions comprising same. A method of treating a subject suffering from an inflammatory condition comprising administering to said subject an antibody or an antigen-binding fragment as described herein.

RELATED APPLICATION(S)

This application claims the benefit of U.S. Provisional Application No.60/737,045, filed Nov. 15, 2005. The entire teachings of the aboveapplication is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Mammals respond to inflammation in part through central nervous systemregulation. One set of responses is through efferent vagus nervesignaling, termed the “cholinergic anti-inflammatory pathway”(Borovikova et al., Vagus nerve stimulation attenuates the systemicinflammatory response to endotoxin. Nature: 405:458-462 (2000)).Stimulation of the efferent vagus nerve or signaling throughα-bungarotoxin-sensitive nicotinic acetylcholine receptors (by, e.g.,acetylcholine) attenuates systemic inflammatory responses and macrophagecytokine synthesis (Bernik, T. R. et al. Pharmacological stimulation ofthe cholinergic anti-inflammatory pathway. J. Exp. Med. 195:781-788(2002); Tracey et al. Mind over immunity. FASEB J. 15:1575-1576 (2001);U.S. patent application Ser. No. 09/855,446).

Nicotinic acetylcholine receptors are a family of ligand-gated,pentameric ion channels. In humans, 16 different subunits (α1-7, α9-10,β1-4, δ, ε, and γ) have been identified that form a large number ofhomo- and hetero-pentameric receptors with distinct structural andpharmacological properties (Lindstrom, 1995; Leonard and Bertrand, 2001;Le Novere and Changeux, 1995). The main known function of this receptorfamily is to transmit signals for the neurotransmitter acetylcholine atneuromuscular junctions and in the central and peripheral nervoussystems (Lindstrom, J. M. Nicotinic acetylcholine receptors. In “HandBook Of Receptors And Channels: Ligand- And Voltage-Gated Ion Channels.”Edited by R. Alan North. CRC Press, Inc. (1995); Leonard, S. andBertrand, D. Neuronal nicotinic receptors: from structure to function.Nicotine & Tobacco Res. 3:203-223 (2001); Le Novere, N. & Changeux, J-P.Molecular evolution of the nicotinic acetylcholine receptor: an exampleof multigene family in excitable cells. J. Mol. Evol. 40:155-172 (1995),Marubio, L. M. and Changeux, J-P. Nicotinic acetylcholine receptorknockout mice as animal models for studying receptor function. Eur. J.Pharmacol. 393:113-121 (2000); Steinlein, O. New functions for nicotineacetylcholine receptors? Behavioural Brain Res. 95:31-35 (1998)).

In a co-pending Published U.S. Patent Application No. 2004/0204355, theidentity of the subunit of an acetylcholine receptor primarilyresponsible for attenuation of inflammatory responses was disclosed tobe the α7 subunit. However, the mechanisms through which the α7 subunitacts and that would facilitate design of novel anti-inflammatorypharmaceutical agents remained unknown.

There is, however, a continued need for pharmaceutically active agentsthat can exploit the cholinergic anti-inflammatory pathway by activatingacetylcholine receptor in a type-specific manner with minimal sideeffects.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that the cholinergicanti-inflammatory pathway can be activated by binding to specificregions of the α7 subunit of a nicotinic receptor. Specifically,Applicants have discovered that the cholinergic anti-inflammatorypathway is activated upon binding to an α7 nicotinic receptor byantibodies cognate to certain fragments of the receptor (Examples 4-7).Based on this discovery, novel antibodies and pharmaceuticalcompositions comprising the same useful for treatment of inflammatoryconditions are disclosed.

In one embodiment, the present invention is an isolated antibody or anantigen binding fragment thereof that specifically binds to a peptideconsisting of an amino acid sequence having at least 80% identity to asequence selected from the group of SEQ ID NO:2, SEQ ID NO:3 and SEQ IDNO:4.

In one embodiment, the present invention is isolated antibody or anantigen-binding fragment thereof that specifically binding to a peptideconsisting of an amino acid sequence having at least 80% identity to SEQID NO:2.

In another embodiment, the present invention is isolated antibody or anantigen-binding fragment thereof that specifically binding to a peptideconsisting of an amino acid sequence having at least 80% identity to SEQID NO:3.

In another embodiment, the present invention is isolated antibody or anantigen-binding fragment thereof that specifically binding to a peptideconsisting of an amino acid sequence having at least 80% identity to SEQID NO:4.

In another embodiment, the present invention is an isolated antibody oran antigen binding fragment thereof which specifically binds to apeptide with an amino acid sequence of SEQ ID NO:2 or a fragmentthereof.

In another embodiment, the present invention is an isolated antibody oran antigen-binding fragment thereof that specifically binds to a peptidewith an amino acid sequence of SEQ ID NO:3 or a fragment thereof.

In a further embodiment, the present invention is an isolated antibodyor an antigen-binding fragment thereof that specifically binds to apeptide with an amino acid sequence of SEQ ID NO:4 or a fragmentthereof.

In one embodiment, the present invention is a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier or diluent and anantibody or an antigen binding fragment thereof that specifically bindsto a peptide consisting of an amino acid sequence having at least 80%identity to a sequence selected from the group of SEQ ID NO:2, SEQ IDNO:3 and SEQ ID NO:4.

In another embodiment, the present invention is a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier or diluentand an antibody or an antigen binding fragment thereof that specificallybinds to a peptide consisting of SEQ ID NO:2 or a fragment thereof.

In one embodiment, the present invention is a method of treating asubject suffering from an inflammatory condition, comprisingadministering to said subject an effective amount of an antibody or anantigen binding fragment thereof. The antibody or the antigen bindingfragment specifically binds to a peptide consisting of an amino acidsequence having at least 80% identity to a sequence selected from thegroup of SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.

In another embodiment, the present invention is a method of treating asubject suffering from an inflammatory condition, comprisingadministering to said subject an effective amount of an antibody or anantigen binding fragment thereof. The antibody or antigen bindingfragment specifically binds to a peptide consisting of an amino acidsequence of SEQ ID NO:2 or a fragment thereof.

Antibodies disclosed herein inhibit pro-inflammatory cytokine productionin vitro (Examples 5 and 6) and protects mice against lethality causedby cecal ligation and puncture (CLP) experimental procedure (Example 7).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the amino acid sequence (SEQ ID NO:1) of human α7subunit of an nicotinic acetylcholine receptor deposited in GenBankunder Accession Number P36544.

FIG. 2 illustrates peptides (SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4)that were used as immunogens to generate antibodies to the α7 subunit.

FIG. 3 is fluorescent micrographs establishing thatα-bungarotoxin-binding nicotinic receptors are clustered on the surfaceof macrophages. Primary human macrophages were stained with FITC-labeledα-bungarotoxin (α-bgt, 1.5 μg/ml) and viewed with a fluorescent confocalmicroscope. Panels A and B show lower magnification micrographs. PanelA: Cells were stained with α-bungarotoxin alone. Panel B: 500 μM ofnicotine was added prior to the addition of α-bungarotoxin. Panels C andD show higher magnification micrographs to reveal the receptor clusters.Panel C: Focus planes were on the inside layers close to the middle(three lower cells) or close to the surface (upper cell) of cells. PanelD: Focus plane was on the upper surface of the cell.

FIG. 4 presents photographs of gels and western blots showing the mRNAand protein expression of α7 and α1 nicotinic receptors in primary humanmarophages. Panel A shows results of RT-PCR with α7-specific primers,generating a 843 bp α7 band. PCR products were verified by sequencing(data not shown). MAC1 and MAC2: macrophages derived from two unrelateddonors. Panel B shows western blots. Cell lysates from PC12 cells orhuman macrophages (MAC) were incubated with either control Sepharosebeads or Sepharose beads conjugated with α-bungarotoxin. The boundproteins were then analyzed by α7-specific polyclonal and monoclonalantibodies as indicated.

FIG. 5 is a photograph of western blot showing binding specificities ofa number of rabbit polyclonal antibodies raised against variousfragments of SEQ ID NO:1. Antibodies 1973-1981 were raised againstvarious fragments of SEQ ID NO:1 spanning positions 23 through 250.Antibody 1918 was raised against a peptide of SEQ ID NO:2; antibody 1921was raised against a peptide of SEQ ID NO:3.

FIG. 6 is a photograph of a western blot showing expression of the α7subunit in the indicated cell lines. The primary antibody used was theantibody 1918 raised against a peptide having the sequence of SEQ IDNO:2.

FIGS. 7A-D are bar plot showing activities of selected antibodies of thepresent invention at different concentrations. Activity is expressed aspercent inhibition of a cytokine release from LPS-stimulated primaryhuman macrophages. FIG. 7A: Inhibition of TNF release at 40 μg/ml of anantibody. FIG. 7B: Inhibition of IL-8 release at 40 μg/ml of anantibody. FIG. 7C: Inhibition of TNF release at 5 ng/ml of an antibody.FIG. 7D: Inhibition of IL-8 release at 5 ng/ml of an antibody.

FIG. 8 is a bar plot showing α7 agonist activities of the 1918 antibodyraised against a fragment of the human α7 subunit of a nicotinicreceptor (SEQ ID NO:1) encompassed in SEQ ID NO:2. Activity is expressedas percent inhibition of TNF release from LPS-stimulated RAW cells.

FIGS. 9A and B illustrate dose dependence of inhibition of TNFα (FIG.9A) and HMGB-1 (FIG. 9B) release from macrophages by α7 antibody 1918,raised against a fragment of the human α7 subunit of a nicotinicreceptor (SEQ ID NO:1) encompassed by SEQ ID NO:2. FIG. 9A is a plot ofpercent inhibition of TNFα release as a function of antibodyconcentration. FIG. 9B is a photograph of a western blot showing thedose-dependent reduction in intensity of the band corresponding toHMGB-1.

FIG. 10 is a plot showing percent survival of mice subjected to cecalligation and puncture (CLP) procedure as a function of a number of dayspost CLP. Two groups of mice are shown: the group treated with antibody1918 (raised against a peptide of SEQ ID NO:2) and the group treated byan irrelevant IgG (control).

FIG. 11A shows immunostaining microphotographs of TNF production in RAW264.7 cells treated either with Ab1918 or an irrelevant IgG cellsfollowing TNF induction by LPS.

FIG. 11B is a plot illustrating dose-dependent inhibition of TNFproduction by Ab1918 in RAW 264.7 cells treated with LPS.

FIG. 12A is a plot illustrating dose-dependent inhibition of HMGB1production by Ab1918 in RAW 264.7 cells treated with LPS.

FIG. 12B shows immunostaining microphotographs of HMGB1 production inRAW 264.7 cells treated either with Ab1918 or an irrelevant IgG cellsfollowing HMGB1 induction by LPS.

FIG. 13 is a bar plot indicating LPS-induced TNF production (as percentof control) in primary human macrophages, following treatment withantisense oligonucleotides to different subunits of acetylcholinereceptors.

FIG. 14A is a bar plot indicating LPS-induced TNF blood serum level inmice following treatment with with Ab1918.

FIG. 14B shows immunostaining microphotographs of spleen tissue sectionstaken from mice treated either with Ab1918 or an irrelevant IgG cellsfollowing TNF induction by LPS.

FIG. 15A is a bar plot indicating LPS-induced HMGB-1 blood serum levelin mice that underwent cecal ligation and puncture, following treatmentwith Ab1918.

FIG. 15B is a plot comparing survival rates of mice that underwent cecalligation and puncture, following treatment with either Ab1918 or anirrelevant IgG.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery of specific regions ofthe α7 subunit, which, upon binding cognate antibodies, stimulate an α7nicotinic acetylcholine receptor. This stimulation, in turn, inhibitssignaling pathways that contribute to inflammatory conditions.Antibodies that have such a stimulatory effect on an α7 nicotinicreceptor are α7 receptor agonists (see also below).

Accordingly, in some embodiments, the present invention is a method oftreatment of an inflammatory condition in a patient. The methodcomprises administering to a subject an effective amount of an antibodyor an antigen binding fragment thereof, wherein the antibody or itsantigen binding fragment stimulates the α7 nicotinic receptor.

As used herein, an “α7 nicotinic receptor” is a nicotinic receptorcomprising an α7 subunit. The receptor can comprise only α7 subunits;alternatively the receptor comprises α7 subunit(s) and subunit(s) fromother nicotinic receptor subtypes. The receptor can be a homopentamer.Alternatively, the receptor can be a heteropentamer. An “α7 subunit” isintended to include all α7 subunit isoforms and/or variants including,but not limited to, the α7 duplicate nicotinic acetylcholine receptor(“dupα7”) described in Villiger et al., Journal of Immunology 126: 86-98(2002) and Gault et al., Genomics 52:173-85 (1998), the splice variantα7-2 described in US 20040152160 and the promoter variant(s) of the α7nicotinic receptor described in U.S. Pat. No. 6,875,606. The relevantteachings of these publications are incorporated herein by reference.

As used herein, an “α7 nicotinic receptor agonist” and a “α7 receptoragonist” is a compound (including an antibody) that binds to a receptorcomprising an α7 subunit, in vivo or in vitro, inducing the receptor toperform its physiological function. As a result of this induction, anicotinic receptor agonist inhibits proinflammatory signaling pathwaysthus alleviating or eliminating inflammation and treating or curinginflammatory conditions.

Antibodies and Antibody-Producing Cells

FIG. 1 shows the amino acid sequence of the human α7 nicotinic receptorsubunit (SEQ ID NO:1). Preferably, the antibody of the present inventionis a selective agonist of the α7 nicotinic receptor.

The antibody of the invention can be polyclonal or monoclonal, and theterm “antibody” is intended to encompass both polyclonal and monoclonalantibodies. The terms polyclonal and monoclonal refer to the degree ofhomogeneity of an antibody preparation, and are not intended to belimited to particular methods of production. The term “antibody” as usedherein also encompasses functional fragments of antibodies, includingfragments of chimeric, humanized, primatized, veneered or single chainantibodies. Functional fragments include antigen-binding fragments whichbind to a mammalian α7 nicotinic receptor. For example, an antibody canbe an IgG or antigen-binding fragment of an IgG. Antibody fragmentscapable of binding to a mammalian α7 nicotinic receptor or fragmentsthereof, include, but are not limited to Fv, Fab, Fab′ and F(ab′)₂fragments. Such fragments can be produced by enzymatic cleavage or byrecombinant techniques. For example, papain or pepsin cleavage cangenerate Fab or F(ab′)₂ fragments, respectively. Other proteases withthe requisite substrate specificity can also be used to generate Fab orF(ab′)₂ fragments. Antibodies can also be produced in a variety oftruncated forms using antibody genes in which one or more stop codonshas been introduced upstream of the natural stop site. For example, achimeric gene encoding a F(ab′)₂ heavy chain fragment can be designed toinclude DNA sequences encoding the CH₁ domain and hinge region of theheavy chain.

Single chain antibodies, and chimeric, humanized or primatized(CDR-grafted), or veneered antibodies, as well as chimeric, CDR-graftedor veneered single chain antibodies, comprising fragments derived fromdifferent species, and the like are also encompassed by the presentinvention and the term “antibody”. The various fragments of theseantibodies can be joined together chemically by conventional techniques,or can be prepared as a contiguous protein using genetic engineeringtechniques. For example, nucleic acids encoding a chimeric or humanizedchain can be expressed to produce a contiguous protein. See, e.g.,Cabilly et al., U.S. Pat. No. 4,816,567; Cabilly et al., European PatentNo. 0,125,023 B1; Boss et al., U.S. Pat. No. 4,816,397; Boss et al.,European Patent No. 0,120,694 B1; Neuberger, M. S. et al., WO 86/01533;Neuberger, M. S. et al., European Patent No. 0,194,276 B1; Winter, U.S.Pat. No. 5,225,539; Winter, European Patent No. 0,239,400 B1; Queen etal., European Patent No. 0 451 216 B1; and Padlan, E. A. et al., EP 0519 596 A1. See also, Newman, R. et al., BioTechnology, 10: 1455-1460(1992), regarding primatized antibody, and Ladner et al., U.S. Pat. No.4,946,778 and Bird, R. E. et al., Science, 242: 423-426 (1988))regarding single chain antibodies.

Humanized antibodies can be produced using synthetic or recombinant DNAtechnology using standard methods or other suitable techniques. Nucleicacid (e.g., cDNA) sequences coding for humanized variable regions canalso be constructed using PCR mutagenesis methods to alter DNA sequencesencoding a human or humanized chain, such as a DNA template from apreviously humanized variable region (see e.g., Kamman, M., et al.,Nucl. Acids Res., 17: 5404 (1989)); Sato, K., et al., Cancer Research,53: 851-856 (1993); Daugherty, B. L. et al., Nucleic Acids Res., 19(9):2471-2476 (1991); and Lewis, A. P. and J. S. Crowe, Gene, 101: 297-302(1991)). Using these or other suitable methods, variants can also bereadily produced. In one embodiment, cloned variable regions can bemutated, and sequences encoding variants with the desired specificitycan be selected (e.g., from a phage library; see e.g., Krebber et al.,U.S. Pat. No. 5,514,548; Hoogenboom et al., WO 93/06213, published Apr.1, 1993).

Antibodies which are specific for a mammalian (e.g., human) α7 nicotinicreceptor can be raised against an appropriate immunogen, such asisolated and/or recombinant human protein of SEQ ID NO:1 or fragmentsthereof (including synthetic molecules, such as synthetic peptides).Antibodies can also be raised by immunizing a suitable host (e.g.,mouse) with cells that express α7 nicotinic receptor, such asmacrophages. In addition, cells expressing a recombinant mammalian α7nicotinic receptor such as transfected cells, can be used as immunogensor in a screen for antibody which binds receptor (see e.g., Chuntharapaiet al., J. Immunol., 152: 1783-1789 (1994); Chuntharapai et al., U.S.Pat. No. 5,440,021).

Preparation of immunizing antigen, and polyclonal and monoclonalantibody production can be performed using any suitable technique. Avariety of methods have been described (see e.g., Kohler et al., Nature,256: 495-497 (1975) and Eur. J. Immunol. 6: 511-519 (1976); Milstein etal., Nature 266: 550-552 (1977), Koprowski et al., U.S. Pat. No.4,172,124; Harlow, E. and D. Lane, 1988, Antibodies: A LaboratoryManual, (Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y.);Current Protocols In Molecular Biology, Vol. 2 (Supplement 27, Summer'94), Ausubel, F. M. et al., Eds., (John Wiley & Sons: New York, N.Y.),Chapter 11, (1991)). Generally, a hybridoma is produced by fusing asuitable immortal cell line (e.g., a myeloma cell line such as SP2/0,P3X63Ag8.653 or a heteromyloma) with antibody producing cells. Antibodyproducing cells can be obtained from the peripheral blood or, preferablythe spleen or lymph nodes, of humans or other suitable animals immunizedwith the antigen of interest. The fused cells (hybridomas) can beisolated using selective culture conditions, and cloned by limitingdilution. Cells which produce antibodies with the desired specificitycan be selected by a suitable assay (e.g., ELISA).

Other suitable methods of producing or isolating antibodies of therequisite specificity (e.g., human antibodies or antigen-bindingfragments) can be used, including, for example, methods which selectrecombinant antibody from a library (e.g., a phage display library), orwhich rely upon immunization of transgenic animals (e.g., mice) capableof producing a repertoire of human antibodies (see e.g., Jakobovits etal., Proc. Natl. Acad. Sci. USA, 90: 2551-2555 (1993); Jalkobovits etal., Nature, 362: 255-258 (1993); Lonberg et al., U.S. Pat. No.5,545,806; Surani et al., U.S. Pat. No. 5,545,807; Lonberg et al.,WO97/13852).

The invention also relates to a bispecific antibody, or functionalfragment thereof (e.g., F(ab′)₂), which binds to a mammalian α7nicotinic receptor and at least one other antigen. (see, e.g., U.S. Pat.No. 5,141,736 (Iwasa et al.), U.S. Pat. Nos. 4,444,878, 5,292,668,5,523,210 (all to Paulus et al.) and U.S. Pat. No. 5,496,549 (Yamazakiet al.)).

In one embodiment, the antibody or antigen-binding fragment of theinvention specifically binds to a fragment of SEQ ID NO:2, SEQ ID NO:3or SEQ ID NO:4. (FIG. 2 illustrates relative positions of thesesequences within an α7 subunit of a human nicotinic receptor.) Thefragment can be 5 to 10 amino acids long, 10 to 15 amino acids long or15 to 19 amino acids long.

Alternatively, the antibody or antigen-binding fragment specificallybinds to SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.

Table 1 lists the above-mentioned amino acid sequences.

TABLE 1 Positions in Amino Acid Sequence SEQ ID NO: 1 SEQ ID NOkelvknynplerpvandsqp  31-50 SEQ ID NO: 2 wkpdillynsaderfdatfh 108-127SEQ ID NO: 3 krserfyecckepypdvtft 204-223 SEQ ID NO: 4

In one embodiment, the antibody or antigen-binding fragment binds afunctional variant of SEQ ID NO:1 as defined below or a fragmentthereof. For example, the antibody or antigen-binding fragment can bindto a peptide that has sequence identity to peptides of SEQ ID NO:2, SEQID NO:3 or SEQ ID NO:4. A peptide against which the antibodies will bindcan share at least about 80% amino acid sequence similarity with apeptide selected from SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4 or afragment thereof, preferably at least about 90% amino acid sequencesimilarity, and more preferably at least about 95% amino acid sequencesimilarity. Methods that can be used to ascertain sequence similarityare described below.

Antibodies of the invention can be identified by a variety of suitablemethods. For example, such an antibody can be identified based upon theability to compete with any of the above-recited antibody for binding tomammalian α7 nicotinic receptor. In another example, the binding of suchan antibody and the binding of an antibody with the same or similarepitopic specificity can be inhibited by a natural peptide or asynthetic peptide. The peptide can comprise five to about fifty aminoacids. Preferably, the peptide comprises ten to about twenty one aminoacids. In still another example, an antibody with the same or similarepitopic specificity as an antibody as recited above can be identifiedusing chimeric receptors (see e.g., Rucker et al., Cell 87:437-446(1996)).

In a particular embodiment, the bispecific antibody, or functionalfragment thereof has the same or similar epitopic specificity as anantibody that binds to a peptide of SEQ ID NO:2, SEQ ID NO:3 or SEQ IDNO:4 and at least one other antibody.

A nonlimiting example of methods for generating antibodies for the α7nicotinic receptor is immunizing a suitable laboratory animal with theα7 receptor or a fragment thereof and isolating the antibodies elicitedby the immunization which bind α7 subunit. Immunization and isolationprocedures are well known to one of ordinary skill in the art.

Selectivity for the α7 nicotinic receptor can be assessed by screeningfor binding to at least one other nicotinic or cholinergic receptor.Antibodies that are found to be selective agonist for the α7 receptormay be further evaluated for their efficacy in inhibiting cytokinerelease and/or in treating one or more of the inflammatory diseasesdescribed herein, e.g., additional in vitro tests or in vivo tests inanimal models.

Antibodies which are agonists can be identified by the proceduresdisclosed herein, for example, by combining the isolated antibodies witha macrophage that has been stimulated to release proinflammatorycytokine, or any other suitable method for assessing α7 receptoractivity. Inhibition of cytokine release is indicative of the agonistactivity.

Methods of identifying an agonist antibody against nicotinic receptorsin general and α7 receptor in particular will be described below. In apreferred method, the binding activity of an agonist anti-α7 antibody ismeasured by the activity (current responses) of Xenopus oocytesexpressing either the α7 receptor subtype or another receptor subtype(e.g., α4β2) following administration of the agonist. Agonists thatresult in greater activation of the α7 receptor subtype are determinedto be α7 selective agonists.

Functional Assays

A composition comprising a mammalian α7 nicotinic receptor or functionalvariant thereof can be used in a binding assay to detect and/or identifyagents that can bind to the receptor, including antibodies of theinvention.

Compositions suitable for use in a binding assay include, for example,cells which naturally express a mammalian α7 nicotinic receptor orfunctional variant thereof and recombinant cells expressing a mammalianα7 nicotinic receptor or functional variant thereof. Compositionssuitable for use in a binding assay also include, membrane preparationswhich comprise a mammalian α7 nicotinic receptor or functional variantthereof. Such membrane preparations can contain natural (e.g., plasmamembrane) or synthetic membranes. Preferably, the membrane preparationis a membrane fraction of a cell that expresses a mammalian α7 nicotinicreceptor or a functional variant thereof.

As used herein, “mammalian α7 nicotinic receptor” refers to a naturallyoccurring or endogenous mammalian protein comprising an α7 subunit of anicotinic receptor and to proteins having an amino acid sequence whichis the same as that of a naturally occurring or endogenous correspondingmammalian α7 subunit protein (e.g., recombinant proteins, syntheticproteins (i.e., produced using the methods of synthetic organicchemistry)). Accordingly, as defined herein, the term includes maturereceptor protein, polymorphic or allelic variants, and other isoforms ofa mammalian α7 subunit (e.g., produced by alternative splicing or othercellular processes), and modified or unmodified forms of the foregoing(e.g., lipidated, glycosylated, unglycosylated). Naturally occurring orendogenous mammalian α7 subunit proteins include wild type proteins suchas mature α7 nicotinic receptor, polymorphic or allelic variants andother isoforms which occur naturally in mammals (e.g., humans, non-humanprimates). Such proteins can be recovered or isolated from a sourcewhich naturally produces mammalian α7 subunit, for example.

“Functional variants” of mammalian α7 nicotinic receptor includefunctional fragments, functional mutant proteins, and/or functionalfusion proteins which can be produce using suitable methods (e.g.,mutagenesis (e.g., chemical mutagenesis, radiation mutagenesis),recombinant DNA techniques). A “functional variant” is a protein orpolypeptide which has at least one function characteristic of amammalian α7 nicotinic receptor protein as described herein, such as abinding activity, a signaling activity and/or ability to stimulate acellular response. Preferred functional variants can bind a ligand(i.e., one or more ligands, such as acetylcholine or an antibody of theinvention).

Generally, fragments of mammalian α7 nicotinic receptor include thosehaving a deletion (i.e., one or more deletions) of an amino acid (i.e.,one or more amino acids) relative to the mature mammalian α7 nicotinicreceptor (such as N-terminal, C-terminal or internal deletions).Fragments in which only contiguous amino acids have been deleted or inwhich non-contiguous amino acids have been deleted relative to maturemammalian α7 nicotinic receptor protein are also envisioned.

Mutant mammalian α7 nicotinic receptors include natural or artificialvariants of a mammalian α7 nicotinic receptor differing by the addition,deletion and/or substitution of one or more contiguous or non-contiguousamino acid residues (e.g., receptor chimeras). Such mutations can occurat one or more sites on a protein, for example a conserved region ornonconserved region (compared to other chemokine receptors or G-proteincoupled receptors), extracellular region, cytoplasmic region, ortransmembrane region.

Fusion proteins encompass polypeptides comprising a mammalian α7 subunitor a variant thereof as a first moiety, linked via a covalent bond(e.g., a peptide bond) to a second moiety not occurring in the mammalianα7 subunit or α7 nicotinic receptor as found in nature. Thus, the secondmoiety can be an amino acid, oligopeptide or polypeptide. The secondmoiety can be linked to the first moiety at a suitable position, forexample, the N-terminus, the C-terminus or internally. In oneembodiment, the fusion protein comprises an affinity ligand (e.g., anenzyme, an antigen, epitope tag, a binding domain) as the first moiety,and a second moiety comprising a linker sequence and human α7 subunit ora fragment thereof. Additional (e.g., third, fourth) moieties can bepresent as appropriate.

In one embodiment, a functional variant of mammalian α7 nicotinicreceptor subunit (e.g., a ligand binding variant) shares at least about80% amino acid sequence similarity with said mammalian α7 subunit,preferably at least about 90% amino acid sequence similarity, and morepreferably at least about 95% amino acid sequence similarity with saidmammalian α7 subunit (e.g., a human α7 subunit (e.g., SEQ ID NO:1)). Inanother embodiment, a functional fusion protein comprises a first moietywhich shares at least about 85% sequence similarity with a mammalian α7subunit, preferably at least about 90% sequence similarity, and morepreferably at least about 95% sequence similarity with a mammalian α7subunit. In another embodiment, a functional mammalian α7 subunit orfunctional variant of a mammalian α7 subunit shares at least about 80%amino acid sequence similarity, preferably at least about 90% amino acidsequence similarity, and more preferably at least about 95% amino acidsequence with a naturally occurring human α7 subunit.

Amino acid sequence similarity can be determined using a suitablesequence alignment algorithm, such as the LASERGENE system (sequenceassembly and alignment software; DNASTAR, Inc., Madison, Wis.), usingthe Clustal method with the PAM 250 residue weight table, a gap penaltyof 10, a gap length penalty of 10 and default parameters (pairwisealignment parameters: ktuple=1, gap penalty=3, window-4 and diagonalssaved=5).

In another embodiment, a functional variant is encoded by a nucleic acidsequence which is different from the naturally-occurring nucleic acidsequence, but which, due to the degeneracy of the genetic code, encodesmammalian α7 subunit or a fragment thereof.

In one embodiment, the method of detecting or identifying an antibodythat binds to a mammalian α7 nicotinic receptor is a competitive bindingassay in which the ability of a test agent (e.g. an antibody) to inhibitthe binding of a reference agent (e.g., a ligand or another antibody ofknown specificity) is assessed. For example, the reference agent can belabeled with a suitable label as described herein, and the amount oflabeled reference agent required to saturate the α7 nicotinic receptorpresent in the assay can be determined. A saturating amount of labeledreference agent and various amounts of a test agent can be contactedwith a composition comprising a mammalian α7 nicotinic receptor orfunctional variant thereof under conditions suitable for binding andcomplex formation determined.

The formation of a complex between either the reference or a test agentand the α7 nicotinic receptor or functional variant thereof or fragmentsthereof including immunogenic peptides as described above can bedetected or measured directly or indirectly using suitable methods. Forexample, the agent can be labeled with a suitable label and theformation of a complex can be determined by detection of the label. Thespecificity of the complex can be determined using a suitable controlsuch as unlabeled agent or label alone. Labels suitable for use indetection of a complex between an agent and a mammalian α7 nicotinicreceptor or functional variant thereof include, for example, aradioisotope, an epitope, an affinity label (e.g., biotin, avidin), aspin label, an enzyme, a fluorescent group or a chemiluminescent group.

With respect to a competitive binding assays used to determine theability of a test agent such as an antibody to bind an α7 nicotinicreceptor, such ability can be reported as the concentration of testagent required for 50% inhibition (IC₅₀ values) of specific binding oflabeled reference agent. Specific binding is preferably defined as thetotal binding (e.g., total label in complex) minus the non-specificbinding. Non-specific binding is preferably defined as the amount oflabel still detected in complexes formed in the presence of excessunlabeled reference agent. Reference agents which are suitable for usein the method include molecules and compounds which specifically bind toa mammalian α7 subunit or a functional variant thereof, for example, aligand of α7 subunit or an antibody. Preferred reference agents areantibodies having a known specificity against the fragments of the α7subunit of a human nicotinic receptor (SEQ ID NO:1) selected from thegroup of SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.

Validation of efficacy of an Ab of the invention can be performed bydetermining whether an antibody inhibits release of a proinflammatorycytokine from a mammalian cell.

These methods preferably involve treating the mammalian cell with anantibody along with an agent that stimulates a proinflammatory cytokinecascade. A preferred agent is bacterial lipopolysaccharide (LPS). Thecompound can be administered to the mammalian cell either before theagent, at the same time as the agent, or after the agent. Preferably,the compound is administered before the agent. See, e.g., U.S. Pat. No.6,610,713, the relevant teachings of which are incorporated herein byreference.

For these methods, the cell can be any cell that can be induced toproduce a proinflammatory cytokine. In preferred embodiments, the cellis an immune cell, for example macrophages, monocytes, or neutrophils.In the most preferred embodiments, the cell is a macrophage.

The proinflammatory cytokine to be measured for inhibition can be anyproinflammatory cytokine that can be induced to be released from thecell. In preferred embodiments, the cytokine is TNF.

Evaluation of the inhibition of cytokine production can be by any meansknown, including quantitation of the cytokine (e.g., with ELISA), or bybioassay, (e.g. determining whether proinflammatory cytokine activity isreduced), or by measurement of the proinflammatory cytokine mRNA. Theskilled artisan could utilize any of these assays without undueexperimentation. See also U.S. Pat. No. 6,610,713, the relevantteachings of which are incorporated herein by reference, for examples ofseveral assays useful in this regard.

These methods can be performed in vivo, where an animal, e.g., a rat, istreated with the compound along with an agent that stimulates aproinflammatory cytokine cascade, and the effect of the agent oninduction of the proinflammatory cytokine cascade is measured, e.g., bymeasuring serum TNF levels. However, due to the relative ease of doingthese types of assays with cells culture rather than with whole animals,the methods are preferably performed in vitro, for example usingmacrophage cultures.

In other embodiments, the invention is directed to methods fordetermining whether an antibody has the ability to inhibit inflammation.In some aspects, these methods comprise determining whether an antibodyis an agonist of the α7 nicotinic receptor. Preferably the methodsfurther comprise determining whether an antibody is selective for α7 bytesting the antibody for its ability to activate at least one othernicotinic receptor. These determinations can be made as previouslydescribed, e.g., by determining whether the antibody inhibits release ofa proinflammatory cytokine from a mammalian cell, preferably amacrophage.

Methods of Therapy

The present invention is directed toward treating cytokine-mediatedinflammatory conditions where the level of cytokine release can bereduced by α7 receptor activation. In preferred embodiments, thecondition is one where the inflammatory cytokine cascade is affectedthrough release of proinflammatory cytokines from a macrophage. Thecondition can be one where the inflammatory cytokine cascade causes asystemic reaction, such as with septic shock. Alternatively, thecondition can be mediated by a localized inflammatory cytokine cascade,as in rheumatoid arthritis.

Nonlimiting examples of conditions which can be usefully treated usingthe present invention include appendicitis, peptic, gastric or duodenalulcers, peritonitis, pancreatitis, acute or ischemic colitis,diverticulitis, epiglottitis, achalasia, cholangitis, cholecystitis,hepatitis, inflammatory bowel disease (including, for example, Crohn'sdisease and ulcerative colitis), enteritis, Whipple's disease, asthma,chronic obstructive pulmonary disease, ileus (including, for example,post-operative ileus), allergy, anaphylactic shock, immune complexdisease, organ ischemia, reperfusion injury, organ necrosis, hay fever,sepsis, septicemia, endotoxic shock, cachexia, hyperpyrexia,eosinophilic granuloma, granulomatosis, sarcoidosis, septic abortion,epididymitis, vaginitis, prostatitis, urethritis, bronchitis, emphysema,rhinitis, cystic fibrosis, pneumonitis, pneumoultramicroscopicsilicovolcanoconiosis, alvealitis, bronchiolitis, pharyngitis, pleurisy,sinusitis, influenza, respiratory syncytial virus, herpes, disseminatedbacteremia, Dengue fever, candidiasis, malaria, filariasis, amebiasis,hydatid cysts, burns, dermatitis, dermatomyositis, sunburn, urticaria,warts, wheals, vasulitis, angiitis, endocarditis, arteritis,atherosclerosis, thrombophlebitis, pericarditis, myocarditis, myocardialischemia, periarteritis nodosa, rheumatic fever, Alzheimer's disease,coeliac disease, congestive heart failure, adult respiratory distresssyndrome, meningitis, encephalitis, multiple sclerosis, cerebralinfarction, cerebral embolism, Guillame-Barre syndrome, neuritis,neuralgia, spinal cord injury, paralysis, uveitis, arthritides,arthralgias, osteomyelitis, fasciitis, Paget's disease, gout,periodontal disease, rheumatoid arthritis, synovitis, myasthenia gravis,thryoiditis, systemic lupus erythematosus, Goodpasture's syndrome,Behcet's syndrome, allograft rejection, graft-versus-host disease, TypeI diabetes, ankylosing spondylitis, Berger's disease, Type II diabetes,Retier's syndrome, or Hodgkins disease.

In one embodiment, the condition is selected from appendicitis, peptic,gastric and duodenal ulcers, peritonitis, pancreatitis, hepatitis,asthma, allergy, anaphylactic shock, organ necrosis, hay fever, sepsis,septicemia, endotoxic shock, cachexia, septic abortion, disseminatedbacteremia, burns, coeliac disease, congestive heart failure, adultrespiratory distress syndrome, chronic obstructive pulmonary disease,rheumatoid arthritis, systemic lupus erythematosis, myocardial ischemia,cerebral infarction, cerebral embolism, spinal cord injury, paralysis,allograft rejection or graft-versus-host disease.

In an alternative embodiment, the condition is selected from the groupconsisting of peritonitis, pancreatitis, sepsis, endotoxic shock, adultrespiratory distress syndrome, chronic obstructive pulmonary disease,rheumatoid arthritis, systemic lupus erythematosis, Crohn's disease,ulcerative colitis, ileus, Alzheimer's disease, burns, myocardialischemia, allograft rejection, asthma, graft-versus-host-disease,congestive heart failure and cystic fibrosis.

These conditions are preferably treated with the antibodies disclosedherein or a combination of an antibody disclosed herein and one or moreadditional agonists of an α7 nicotinic receptor. Suitable additionalagonists are disclosed, for example, in a co-pending U.S. patentapplication Ser. No. 10/729,427. The relevant teachings of U.S. patentapplication Ser. No. 10/729,427 are incorporated herein by reference.

Modes of Administration

The route of administration of the α7 receptor agonist depends on thecondition to be treated. For example, intravenous injection may bepreferred for treatment of a systemic disorder such as septic shock, andoral administration may be preferred to treat a gastrointestinaldisorder such as a gastric ulcer.

According to the method, one or more antibodies of the present inventioncan be administered to the subject by an appropriate route, either aloneor in combination with another drug. An effective amount of an agent(i.e. a α7 receptor agonist antibody or antigen-binding fragmentthereof) is administered. An “effective amount” is an amount sufficientto achieve the desired therapeutic or prophylactic effect, under theconditions of administration, such as an amount sufficient forinhibition of an inflammatory response and alleviating or curing aninflammatory condition. The agents can be administered in a single doseor multiple doses. The dosage can be determined by methods known in theart and is dependent, for example, upon the particular agent chosen, thesubject's age, sensitivity and tolerance to drugs, and overallwell-being. Suitable dosages for antibodies can be from about 0.01 mg/kgto about 100 mg/kg body weight per treatment.

A variety of routes of administration are possible including, forexample, oral, dietary, topical, transdermal, rectal, parenteral (e.g.,intravenous, intraarterial, intramuscular, subcutaneous injection,intradermal injection), and inhalation (e.g., intrabronchial, intranasalor oral inhalation, intranasal drops) routes of administration,depending on the agent and disease or condition to be treated.Administration can be local or systemic as indicated. The preferred modeof administration can vary depending upon the particular agent (α7receptor agonist antibody or an antigen-binding fragment thereof)chosen, and the particular condition (e.g., disease) being treated.Intravenous, oral or parenteral administration are preferred.

The agent can be administered as a neutral compound or as apharmaceutically acceptable salt. Salts of compounds containing an amineor other basic group can be obtained, for example, by reacting with asuitable organic or inorganic acid, such as hydrogen chloride, hydrogenbromide, acetic acid, perchloric acid and the like. Compounds with aquaternary ammonium group also contain a counteranion such as chloride,bromide, iodide, acetate, perchlorate and the like. Salts of compoundscontaining a carboxylic acid or other acidic functional group can beprepared by reacting with a suitable base, for example, a hydroxidebase. Salts of acidic functional groups contain a countercation such assodium, potassium and the like.

As used herein, a “pharmaceutically acceptable salt” of a disclosedcompound is an ionic bond-containing product of reacting a compound ofthe invention with either an acid or a base, suitable for administeringto a subject. For example, an acid salt of a compound containing anamine or other basic group can be obtained by reacting the compound witha suitable organic or inorganic acid, such as hydrogen chloride,hydrogen bromide, acetic acid, perchlioric acid and the like. Otherexamples of such salts include hydrochlorides, hydrobromides, sulfates,methanesulfonates, nitrates, maleates, acetates, citrates, fumarates,tartrates (e.g. (+)-tartrates, (−)-tartrates or mixtures thereofincluding racemic mixtures), succinates, benzoates and salts with aminoacids such as glutamic acid. Salts can also be formed with suitableorganic bases when the compound comprises an acid functional group suchas —COOH or —SO₃H. Such bases suitable for the formation of apharmaceutically acceptable base addition salts with compounds of thepresent invention include organic bases that are nontoxic and strongenough to react with the acid functional group. Such organic bases arewell known in the art and include amino acids such as arginine andlysine, mono-, di-, and triethanolamine, choline, mono-, di-, andtrialkylamine, such as methylamine, dimethylamine, and trimethylamine,guanidine, N-benzylphenethylamine, N-methylglucosamine,N-methylpiperazine, morpholine, ethylendiamine,tris(hydroxymethyl)aminomethane and the like.

The agent can be administered to the individual as part of apharmaceutical composition for modulation of nicotinic receptor functioncomprising an inhibitor of such function and a pharmaceuticallyacceptable carrier.

As used herein, a “pharmaceutical composition” is a formulationcomprising the disclosed antibodies and a pharmaceutically acceptablediluent or carrier, in a form suitable for administration to a subject.Suitable pharmaceutically acceptable carriers include inert solidfillers or diluents and sterile aqueous or organic solutions.Formulation will vary according to the route of administration selected(e.g., solution, emulsion, capsule). Suitable pharmaceutical carrierscan contain inert ingredients which do not interact with the promoter(agonist) or inhibitor (antagonist) of nicotinic receptor function.Standard pharmaceutical formulation techniques can be employed, such asthose described in Remington's Pharmaceutical Sciences, Mack PublishingCompany, Easton, Pa. Suitable pharmaceutical carriers for parenteraladministration include, for example, sterile water, physiologicalsaline, bacteriostatic saline (saline containing about 0.9% mg/ml benzylalcohol), phosphate-buffered saline, Hank's solution, Ringer's-lactateand the like. Methods for encapsulating compositions (such as in acoating of hard gelatin or cyclodextran) are known in the art (Baker, etal., “Controlled Release of Biological Active Agents”, John Wiley andSons, 1986). For inhalation, the agent can be solubilized and loadedinto a suitable dispenser for administration (e.g., an atomizer,nebulizer or pressurized aerosol dispenser).

The pharmaceutical composition can be in bulk or in unit dosage form.The unit dosage form can be in any of a variety of forms, including, forexample, a capsule, an IV bag, a tablet, a single pump on an aerosolinhaler, or a vial. The quantity of active ingredient (i.e., aformulation of the disclosed compound or salts thereof) in a unit doseof composition is an effective amount and may be varied according to theparticular treatment involved. It may be appreciated that it may benecessary to make routine variations to the dosage depending on the ageand condition of the patient. The dosage will also depend on the routeof administration.

As used herein, a “subject” includes mammals, e.g., humans, companionanimals (e.g., dogs, cats, birds and the like), farm animals (e.g.,cows, sheep, pigs, horses, fowl and the like) and laboratory animals(e.g., rats, mice, guinea pigs and the like). In a preferred embodimentof the disclosed methods, the subject is human.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell culture, molecular biology,microbiology, cell biology, and immunology, which are well within theskill of the art. Such techniques are fully explained in the literature.See, e.g., Sambrook et al., 1989, “Molecular Cloning: A LaboratoryManual”, Cold Spring Harbor Laboratory Press; Ausubel et al. (1995),“Short Protocols in Molecular Biology”, John Wiley and Sons; Methods inEnzymology (several volumes); Methods in Cell Biology (several volumes),and Methods in Molecular Biology (several volumes).

Preferred embodiments of the invention are described in the followingExamples. Other embodiments within the scope of the claims herein willbe apparent to one skilled in the art from consideration of thespecification or practice of the invention as disclosed herein. It isintended that the specification, together with the examples, beconsidered exemplary only, with the scope and spirit of the inventionbeing indicated by the claims which follow the Example.

Exemplificaton Example 1 Macrophages Express Nicotinic Receptor α7Subunit Example Summary

We previously reported that the α7 subunit of a nicotinic receptor isrequired for acetylcholine-mediated inhibition of macrophage TNFrelease. (See co-pending U.S. patent application Ser. No. 10/729,427.)As summarized below, α-bungarotoxin bound to discrete receptor clustersexpressed on the surface of primary human macrophages and immunoblottingof proteins isolated by adherence to α-bungarotoxin-conjugated beadswith α7-specific antibodies thus confirming the identity of theα-bungarotoxin-binding receptor subunit as the α7 subunit.

Results and Discussion

Primary human macrophages were labeled with FITC-α-bungarotoxin, apeptide antagonist that binds to a subset of acetylcholine receptors.Strong binding of α-bungarotoxin was observed on the macrophage surface(FIG. 3A). Nicotine pretreatment markedly reduced the intensity ofbinding (FIG. 3B). At neuro-muscular junctions and the neuronalsynapses, nicotinic receptors form receptor aggregates or clusters thatfacilitate fast signal transmission. Discrete clusters of α-bungarotoxinbinding can be clearly observed under higher magnification on thesurface of macrophages, especially concentrated on the surface of thecell body (FIG. 3C, D).

To date, α1, α7 and α9 are the α-bungarotoxin-binding nicotinic receptorsubunits known in human cells (Lindstrom, J. M. Nicotinic acetylcholinereceptors. In “Hand Book Of Receptors And Channels: Ligand- AndVoltage-Gated Ion Channels.” Edited by R. Alan North. CRC Press, Inc.(1995); Leonard, S. and Bertrand, D. Neuronal nicotinic receptors: fromstructure to function. Nicotine & Tobacco Res. 3:203-223 (2001)). α1together with β1, δ and either ε (adult) or γ (fetal) subunits, formsheteropentameric nicotinic receptors that regulate muscle contraction;α7 and α9 can each form homopentameric nicotinic receptors. To determineif these receptor subunits are expressed in macrophages, we isolated RNAfrom primary human macrophages differentiated in vitro from peripheralblood mononuclear cells (PBMC) and performed RT-PCR analyses. Toincrease the sensitivity and specificity of the experiments, weconducted two rounds of PCR after reverse transcription, using nestedprimers specific to each subunit. The identities of the PCR productswere confirmed by sequencing. The expression of α1, α10, (data notshown) and α7 (FIG. 4A) mRNA was detected in human macrophages derivedfrom unrelated blood donors. The same RT-PCR strategy did not detect theexpression of α9 subunit mRNA in macrophages (data not shown).

The protein expression of α1 and α7 subunits was next examined bywestern blotting. The α7 specific antibody recognized a clear band withan apparent molecular weight of about 55 kD (similar to the molecularweight for α7 protein reported by Peng, X. et al. Human α7 acetylcholinereceptor: cloning of the α7 subunit from the SH-SY5Y cell line anddetermination of pharmacological properties of native receptors andfunctional α7 homomers expressed in Xenopus oocytes. Mol. Pharmacol.45:546-554 (1994)) from both differentiated primary macrophages and fromundifferentiated PBMCs (data not shown). α1 protein expression wasdown-regulated to undetectable levels during in vitro differentiation ofPBMC to macrophages (data not shown). The δ subunit, a necessarycomponent of the α1 heteropentameric nicotinic acetylcholine receptor,could not be detected by this nested RT-PCR strategy (data not shown).To confirm that the positive signals in macrophages represented α7nicotinic receptor that binds α-bungarotoxin, we usedα-bungarotoxin-conjugated beads to pull-down proteins prepared fromeither human macrophages or PC12 cells (rat pheochromocytoma cells,which have been shown to express α7 homopentamer). Retained proteinswere analyzed by western blotting using polyclonal or monoclonal α7specific antibodies that recognized both human and rat α7 protein (thehuman and rat α7 proteins contain the same number of amino acids and are94% identical). The results clearly showed that the human macrophagesexpress α-bungarotoxin-binding α7 protein with apparent molecular weightthat is similar to α7 subunit in PC12 cells (FIG. 4B). The identity ofthe macrophage α7 subunit was confirmed by cloning of the full-lengthmacrophage-expressed α7 by RT-PCR methods. The full-length nicotinicacetylcholine α7 subunit in macrophages contains exons 1 to 10,identical to the nicotinic acetylcholine α7 subunit expressed in neurons(Gault, J. et al. Genomic organization and partial duplication of thehuman α7 neuronal nicotinic acetylcholine receptor gene (CHRNA7).Genomics 52:173-185 (1998)). Together, these data identify the nicotinicacetylcholine α7 subunit as the α-bungarotoxin-binding receptorexpressed on the surface of human macrophages.

Example 2 Preparation of Rabbit Polyclonal Antibody Against α7 NicotinicReceptors

Polyclonal antibodies against the extracellular domain and specificpeptides of the α7 nicotinic receptor were generated in rabbitsaccording to the 118 day protocol for Custom Polyclonal AntibodyProduction (Covance Research Products, Inc., Denver, Pa.). Briefly, thepeptides were diluted with sterile saline and combined with an equalvolume of Freund's Complete Adjuvant. 0.125 mg antigen (antigen includesthe peptides described below and GST N-terminal extracellular domain) inFreund's Incomplete Adjuvant were injected six times subcutaneously intorabbits at weeks 3, 6, 9, 12 and 15 after pre-bleed at Day 0. Blood wascollected from the ear vein 10 days after injection at weeks 9, 12 and15. Sera from the rabbits were assayed for titer by Western blotting.IgG was purified from anti-α7 antiserum using Protein A agaroseaccording to manufacturer's instructions [Sigma, St. Louis, Mo.].

The peptide KELVINYNPLERPVANDSOP (SEQ ID NO: 2, corresponding to aminoacids 31-50 of SEQ ID NO:1) was injected into two different rabbits inorder to generate antibodies 1918 and 1920. The peptideWKPDILLYNSADERFDATFH (SEQ ID NO: 3, corresponding to amino acids 108-127of SEQ ID NO:1) was injected in two different rabbits in order togenerate antibodies 1921 and 1922. The peptide KRSERFYECCKEPYPDYTYT (SEQID NO:4, corresponding to amino acids 204-223 of SEQ ID NO:1) wasinjected into two different rabbits in order to generate antibodies 1923and 1924. Amino acids 1-227 (corresponding to the extracellular domainof α7), was expressed as a GST-fusion protein in E. coli. The GST-fusionprotein was purified and was injected into ten different rabbits togenerate antibodies 1973 to 1982.

Results are presented in FIG. 5, which shows a photograph of a westernblot showing binding specificities of a number of rabbit polyclonalantibodies raised against various fragments of SEQ ID NO:1. Antibodies1973-1981 were raised against various fragments of SEQ ID NO:1 spanningpositions 23 through 250. Antibody 1918 was raised against a peptide ofSEQ ID NO:2; antibody 1921 was raised against a peptide of SEQ ID NO:3.

Example 3 Expression of α7 Subunit of a Nicotinic Receptor in Cell Lines

The expression of the α7 subunit in cell lines and primary humanmacrophages was screened by Western blot using antibody 1918.

Murine macrophage-like RAW 264.7, Jurkat cell, Hela cell, U937cell andPC12 cell were obtained from ATCC (American Type Culture Collection,Rockville, Md.). Cells were cultured in RPMI 1640 or DMEM medium,supplemented with 10% heat-inactivated FBS, 2 mM glutamine, 1×penicillin and streptomycin in a humidified incubator with 5% CO₂ and95% room air. Primary human macrophages were isolated from blood ofthree donors.

Cell lysates were prepared by incubating the cells with cold lysisbuffer (1% Brij 97, 50 mM Tris, pH7.5, 150 mM sodium chloride, 1 mMEDTA, 1 mg/ml each of leupeptin and pepstatin, 1 mM PMSF and 10 mMsodium fluoride). Each cell lysate (2×10⁵ cell equivalents) weresubjected to SDS-PAGE and immunoblotted with antibody 1918. The signalwas detected using an ECL kit as instructed by the manufacturer(Amersham Life Science, Inc., Arlington Heights, Ill.).

The results of this experiment are presented in FIG. 6.

Example 4 Antibodies Raised Against a Fragment of α7 Subunit of aNicotinic Receptor Comprising SEQ ID NO:2 Inhibit TNFα and IL-8Production in LPS-Stimulated Macrophages

Human macrophage cultures were prepared as follows. Buffy coats werecollected from the blood of healthy individual donors. Primary bloodmononuclear cells were isolated by density-gradient centrifugationthrough Ficoll/Hypaque (Pharmacia, N.J.), suspended (8×10⁶ cells/ml) inRPMI 1640 medium supplemented with 10% heat inactivated human serum(Gemini Bio-Products, Inc., Calabasas, Calif.), and seeded in flasks(PRIMARIA; Beckton and Dickinson Labware, Franklin Lakes, N.J.). Afterincubation for 2 hours at 37° C., adherent cells were washedextensively, treated briefly with 10 mM EDTA, detached, resuspended (10⁶cells/ml) in RPMI medium (10% human serum), supplemented with humanmacrophage colony stimulating factor (MCSF; Sigma Chemical Co., St.Louis, Mo.; 2 ng/ml), and seeded onto 24-well tissue culture plates(PRIMARIA; Falcon) (10⁶ cells/well). Cells were allowed to differentiatefor 7 days in the presence of mCSF. On day 7 the cells were washed 3times with 1× Dulbecco's phosphate buffered saline (PBS, GibcoBRL, LifeTechnologies, Rockville, Md.), fresh medium devoid of mCSF was added,and experiments performed as indicated.

Primary human macrophage cultures were established by incubating humanperipheral blood mononuclear cells in the presence of macrophage colonystimulating factor (MCSF; Sigma Chemical Co., St. Louis, Mo.). Thesecells were used in experiments to determine the effects of theantibodies of the present invention on TNFα and IL-8 levels inmacrophage cultures conditioned by exposure to LPS for 4 hours.

In these experiments, antibodies 1918 (raised against the peptide of SEQID NO:2), 1921 (raised against the peptide of SEQ ID NO:3) as well asantibodies 1924, 1973, 1974 and 1976 (raised against various fragmentsof SEQ ID NO:1) as well as control (irrelevant IgG antibody) was addedto human macrophage cultures at the concentrations of either 40 μg/ml or5 μg/ml as indicated for thirty minutes (FIGS. 7A-D). LPS was addedthirty minutes later (5 ng/ml), and conditioned supernatants collectedafter 2.5 hours of stimulation for subsequent analysis by enzyme-linkedimmunosorbent assay (ELISA). All the experimental conditions wereperformed in triplicate. Data from nine separate macrophage preparationsare shown as Mean±SEM; n=9.

As can be seen from FIGS. 7A-D, all tested antibodies showed inhibitoryactivity (i.e. agonist activity with respect to the acetylcholinereceptor) activity at 40 μg/ml when compared to an irrelevant antibodycontrol. Even at 5 ng/ml, antibodies 1918, 1921 and 1924 showedsignificant inhibition of TNFα and IL-8 release compared to the control.

Example 5 Antibodies Raised Against a Fragment of α7 Subunit of aNicotinic Receptor Comprising SEQ ID NO:2 Suppress TNF Release fromLPS-Stimulated RAW Cells

Murine RAW 264.7 macrophage-like cells (American Type Tissue CultureCollection, Rockville, Md., USA) were grown under DMEM supplemented with10% fetal bovine serum, penicillin and streptomycin. The cells wereseeded in 24-well tissue culture plates in Opti-MEM 1 medium and used at90% confluence. As indicated, cells were treated with LPS (5 ng/ml) for2.5 hours. As indicated, the cells were pre-treated with 40 ug/ml ofantibody 1918 (raised against the peptide of SEQ ID NO:2) for thirtyminutes. Supernatants were collected and TNF concentration was measuredby mouse ELISA kit (R&D Systems Inc., Minneapolis, Minn.).

The results are shown in FIG. 8, which clearly shows that antibody 1918inhibited TNFα production in RAW cells.

Example 6 Antibodies Raised Against a Fragment of U7 Subunit of aNicotinic Receptor Comprising SEQ ID NO:2 Suppress TNFα and HMGB-1Release from LPS-Stimulated Macrophages in a Dose-Dependent Manner

Macrophages were grown as described in Example 4 above.

In the experiments in which the inhibitory effect of antibody 1918(raised against the peptide of SEQ ID NO:2) on TNFα release wasdetermined, the antibody was added at 0, 2.5, 5, 10, 20, 40, 80, and 160μg/ml. In the experiments in which the inhibitory effect of antibody1918 on HMGB-1 release was determined, the antibody was added at 0, 10,50 and 100 ng/ml.

Thirty minutes after the addition of antibody 1918, the cultures weretreated with LPS at a concentration of 5.0 ng/ml. Culture medium wascollected after 18 hours. The culture medium was concentrated with aCentricon™ 10 filter. TNFα levels was analyzed using a human ELISA kit(R&D Systems Inc., Minneapolis, Minn.). HMGB-1 level was analyzed bywestern blot using anti-HMGB1 polyclonal antisera as described in U.S.Pat. No. 6,303,321, the relevant teachings of which are incorporatedherein by reference. The results of the TNFα inhibition are presented inFIG. 9A as percent stimulation of LPS treatment alone. The results ofHMGB-1 inhibition are presented in FIG. 9B as the progressive decreasein band intensity.

FIGS. 9A and 9B clearly shows that antibody 1918 inhibited release ofTNFα and HMGB-1 in a dose-dependent manner.

Example 7 Agonist Antibody Raised Against a Peptide of SEQ ID NO:2Protects Mice Against Lethality Caused by CLP

Cecal Ligation and Puncture (CLP) was performed as described in Fink andHeard, J. of Surg. Res. 49:186-196 (1990), Wichman et al., Crit. CareMed. 26:2078-2086 (1998) and Remick et al., Shock 4:89-95 (1995).Briefly, Balb/c mice were anesthetized with 75 mg/kg Ketamine (FortDodge, Fort Dodge, Iowa) and 20 mg/kg of xylazine (Bohringer Ingelheim,St. Joseph, Mo.) intramuscularly. A midline incision was performed, andthe cecum was isolated. A 6-0 prolene suture ligature was placed at alevel 5.0 mm from the cecal tip away from the ileocecal valve.

The ligated cecal stump was then punctured once with a 22-gauge needle,without direct extrusion of stool. The cecum was then placed back intoits normal intra-abdominal position. The abdomen was then closed with arunning suture of 6-0 prolene in two layers, peritoneum and fasciaseparately to prevent leakage of fluid. All animals were resuscitatedwith a normal saline solution administered subcutaneously at 20 ml/kg ofbody weight. Each mouse received a subcutaneous injection of imipenem(0.5 mg/mouse) (Primaxin, Merck & Co., Inc., West Point, Pa.) 30 minutesafter the surgery. Animals were then allowed to recuperate.

Mice were treated with either antibody 1918 (raised against a peptide ofSEQ ID NO:2) at 180 mg/mouse twice daily or a control IgG antibody at180 mg/mouse twice daily. The agonist and the control antibodies wereadministered intraperitoneally (i.p.) twice a day for the indicatednumber of days. Mortality was monitored daily for fourteen days aftersurgery. The results are presented in FIG. 10, which shows thepercentage of surviving animals following treatment with either agonist1918 antibody, or irrelevant control. As shown in FIG. 10, at 7 dayspost-CLP, about 10% of mice treated with IgG control antibody survivedwhereas about 50% of mice treated with antibody 1918 survived. Theseresults demonstrate that agonist antibodies raised against a specificregion of the α7 subunit significantly improved survival in the murineCLP model of sepsis.

Example 8 Antibody 1918 Inhibits Endotoxin-Induced TNF Release in RAW264.7 Cell

RAW cells were exposed to LPS in the presence IgG or Ab1918 (20 mg/ml).TNF in the cell was analyzed 1.5 hours later by immunostaining withanti-TNF antibodies. The results are presented in FIG. 11A. As can beseen from the diminished fluorescence (the lower right panel), treatmentwith Ab1918 significantly reduces the level of TNF induced by LPStreatment.

FIG. 11B demonstrates dose-dependent inhibition of TNF release byAb1918. Primary human macrophages were exposed to LPS in the presenceIgG or Ab1918 (1-180 mg/ml) and TNF in the media was analyzed 2 hourlater by ELISA.

Example 9 Antibody 1918 Inhibits LPS-Induced HMGB1 Release from RAW264.7

RAW cells were exposed to LPS in the presence or absence of Ab1918(5-100 mg/ml). Secreted HMGB1 was determined from media 24 hours laterby Western blot. Results are presented in FIG. 12A, which showsdose-dependent inhibition of HMGB1 production by Ab1918, and FIG. 12B,which shows a significant reduction in LPS-induced HMGB1 production byRAW 264.7 cells following treatment with Ab1918.

Example 9 Antisense Oligonucleotides to α7 Subunit of AcetylcholineReceptor Inhibit the Effect of Ab1918 on TNF Release from PrimaryMacrophages

To show that Ab1918 inhibits TNF release by binding the α7 subunit ofacetylcholine (Ach) receptor, TNF release from LPS-stimulated primaryhuman macrophages pretreated with antisense oligonucleotides todifferent subunits of Ach receptors was measured. The α7 subunit wasdetected by western blot using Ab1918 and TNF in the media was analyzedby ELISA. Results are presented in FIG. 13 which shows that when α7subunit synthesis was inhibited by antisense oligonucleotides, treatmentby Ab1918 did not reduce the TNF production, which remained on the levelcomparable to those of no antibody or irrelevant antibody treatments.

Example 10 Antibody 1918 Suppresses Systemic TNF in Endotoxemic Mice

Mice (n=11 per group) were treated introperitoneally (i.p.) with IgG(controls) or Ab1918 (7.2 mg/kg) 12 hour before LPS (7.0 mg/kg, i.p.)administration. Serum TNF levels were analyzed by ELISA in bloodobtained 1.5 hours later (*P<0.05 versus controls). The results arepresented in FIG. 14(A), which shows reduction in serum TNF levelsfollowing administration of Ab1918.

TNF levels in the spleen were analyzed 1.5 hours post LPS administrationby immunostaining with anti-TNF antibodies. The results are presented inFIG. 14(B), which shows a significant reduction in LPS-induced TNFlevels in spleens of mice treated with Ab1918.

Example 11 Ab1918 Suppresses Systemic HMGB1 Level and Improves Survivalof mice with Cecal Ligation and Puncture-Induced Severe Sepsis

Mice were treated i.p. with IgG (controls) or Ab1918 (n=7 per group)twice daily (7.2 mg/kg) for 2 days after cecal ligation and puncture(CLP). Serum HMGB1 was analyzed by Western blot (*p<0.05 versuscontrols). The results are presented in FIG. 15A, which shows asignificant reduction in serum HMGB1 levels following treatment withAb1918.

In survival experiments, mice (n=24) were treated with IgG or Ab1918(7.2 mg/kg), for 3 days, beginning 24 hours after surgery (*P<0.0008versus controls). The results are presented in FIG. 15B, which showssignificant improvement in survival rates of mice treated with Ab1918compared to mice that received sham treatment with an irrelevant IgG.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. An isolated antibody or an antigen-binding fragment thereof thatspecifically binds to a peptide consisting of an amino acid sequencehaving at least 80% identity to a sequence selected from the groupconsisting of SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.
 2. The antibodyor antigen-binding fragment of claim 1 wherein the antibody or fragmentspecifically binds to a peptide consisting of an amino acid sequencehaving at least 80% identity to SEQ ID NO:2.
 3. The antibody orantigen-binding fragment of claim 1 wherein the antibody or fragmentspecifically binds to a peptide consisting of an amino acid sequencehaving at least 80% identity to SEQ ID NO:3.
 4. The antibody orantigen-binding fragment of claim 1 wherein the antibody or fragmentspecifically binds to a peptide consisting of an amino acid sequencehaving at least 80% identity to SEQ ID NO:4.
 5. The antibody or antigenbinding fragment of claim 1 wherein the antibody or fragmentspecifically binds to a peptide consisting of an amino acid sequenceselected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, a fragment of SEQ ID NO:2, a fragment of SEQ ID NO:3 and afragment of SEQ ID NO:4.
 6. The antibody or antigen-binding fragment ofclaim 1 wherein said antibody or fragment is human, humanized orchimeric.
 7. The antibody or antigen-binding fragment of claim 1 whereinsaid antibody or fragment is monoclonal.
 8. The antibody orantigen-binding fragment of claim 1 wherein said antibody or fragment ispolyclonal.
 9. The antigen-binding fragment of claim 1 wherein saidantigen-binding fragment is selected from the group consisting of an Fabfragment, an Fab′ fragment, an F(ab)′₂ fragment and an Fv fragment. 10.An isolated antibody or an antigen-binding fragment thereof thatspecifically binds to a peptide consisting of SEQ ID NO:2 or a fragmentthereof.
 11. An isolated antibody or an antigen-binding fragment thereofthat specifically binds to a peptide consisting of SEQ ID NO:3 or afragment thereof.
 12. An isolated antibody or an antigen-bindingfragment thereof that specifically binds to a peptide consisting of SEQID NO:4 or a fragment thereof.
 13. A pharmaceutical compositioncomprising a pharmaceutically acceptable carrier or diluent and anantibody or an antigen-binding fragment thereof that specifically bindsto a peptide consisting of an amino acid sequence having at least 80%identity to a sequence selected from the group of SEQ ID NO:2, SEQ IDNO:3 and SEQ ID NO:4.
 14. The composition of claim 13 wherein saidantibody or antigen-binding fragment specifically binds to a peptideconsisting of an amino acid sequence selected from the group consistingof SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, a fragment of SEQ ID NO:2, afragment of SEQ ID NO:3 and a fragment of SEQ ID NO:4.
 15. Thecomposition of claim 13 wherein said antibody or fragment is human,humanized or chimeric.
 16. The composition of claim 13 wherein saidantibody or antigen-binding fragment is monoclonal.
 17. The compositionof claim 13 wherein said antibody or antigen-binding fragment ispolyclonal.
 18. The composition of claim 13 wherein said antigen-bindingfragment is selected from the group consisting of an Fab fragment, anFab′ fragment, an F(ab)′₂ fragment and an Fv fragment.
 19. Apharmaceutical composition comprising a pharmaceutically acceptablecarrier or diluent and an antibody or an antigen-binding fragmentthereof that specifically binds to a peptide consisting of SEQ ID NO:2or a fragment thereof.
 20. A pharmaceutical composition comprising apharmaceutically acceptable carrier or diluent and an antibody orantigen-binding fragment thereof that specifically binds to a peptideconsisting of SEQ ID NO:3 or a fragment thereof.
 21. A pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier or diluentand an antibody or antigen-binding fragment thereof that specificallybinds to a peptide consisting of SEQ ID NO:4 or a fragment thereof. 22.A method of treating a subject suffering from an inflammatory condition,comprising: administering to said subject an effective amount of anantibody or an antigen-binding fragment thereof, wherein the antibody orthe antigen-binding fragment specifically binds to a peptide consistingof an amino acid sequence having at least 80% identity to a sequenceselected from the group of SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4. 23.The method of claim 22 wherein said antibody or fragment specifically $binds to a peptide consisting of an amino acid sequence selected fromthe group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, afragment of SEQ ID NO:2, a fragment of SEQ ID NO:3 and a fragment of SEQID NO:4.
 24. The method of claim 22 wherein said condition is selectedfrom the group consisting of appendicitis, peptic, gastric and duodenalulcers, peritonitis, pancreatitis, Crohn's disease, ulcerative colitis,ileus, burns, Alzheimer's disease, epiglottitis, achalasia, cholangitis,cholecystitis, hepatitis, Whipple's disease, asthma, allergy,anaphylactic shock, immune complex disease, organ ischemia, reperfusioninjury, organ necrosis, hay fever, sepsis, septicemia, endotoxic shock,cachexia, hyperpyrexia, eosinophilic granuloma, granulomatosis,sarcoidosis, septic abortion, epididymitis, vaginitis, prostatitis,urethritis, bronchitis, emphysema, rhinitis, cystic fibrosis,pneumonitis, pneumoultramicroscopic silicovolcanoconiosis, alvealitis,bronchiolitis, pharyngitis, pleurisy, sinusitis, influenza, respiratorysyncytial virus infection, herpes infection, HIV infection, hepatitis Bvirus infection, hepatitis C virus infection, disseminated bacteremia,Dengue fever, candidiasis, malaria, filariasis, amebiasis, hydatidcysts, burns, vasulitis, angiitis, endocarditis, arteritis,atherosclerosis, thrombophlebitis, pericarditis, myocarditis, myocardialischemia, periarteritis nodosa, rheumatic fever, coeliac disease,congestive heart failure, adult respiratory distress syndrome, chronicobstructive pulmonary disease, meningitis, encephalitis, neuritis,neuralgia, spinal cord injury, paralysis, uveitis, arthritides,arthralgias, osteomyelitis, fasciitis, Paget's disease, gout,periodontal disease, rheumatoid arthritis, synovitis, myasthenia gravis,thryoiditis, systemic lupus erythematosus, Goodpasture's syndrome,Behcets's syndrome, allograft rejection, graft-versus-host disease,ankylosing spondylitis, Berger's disease, Retier's syndrome, andHodgkins disease.
 25. The method of claim 24 wherein the condition isselected from the group consisting of peritonitis, pancreatitis, sepsis,endotoxic shock, Crohn's disease, ulcerative colitis, ileus, burns,Alzheimer's disease, adult respiratory distress syndrome, chronicobstructive pulmonary disease, rheumatoid arthritis, systemic lupuserythematosis, myocardial ischemia, allograft rejection, asthma,graft-versus-host-disease, congestive heart failure and cystic fibrosis.26. The method of claim 22, wherein the mammal is a human.
 27. Themethod of claim 22 wherein said antibody or antigen-binding fragment ishuman, humanized or chimeric.
 28. The method of claim 22 wherein saidantibody or fragment is monoclonal.
 29. The method of claim 22 whereinsaid antibody or fragment is polyclonal.
 30. The method of claim 22wherein said antigen-binding fragment is selected from the groupconsisting of an Fab fragment, an Fab′ fragment, an F(ab)′₂ fragment andan Fv fragment.
 31. A method of treating a subject suffering from aninflammatory condition, comprising administering to said subject aneffective amount of an antibody or an antigen binding fragment thereof,wherein said antibody or antigen-binding fragment specifically binds apeptide consisting of SEQ ID NO:2 or a fragment thereof.